CN111579703A - LC-MS/MS-based method for detecting and analyzing degradation products of bilirubin in biological sample - Google Patents

Abstract

The invention discloses a method for determining the contents of three bilirubin oxidation products BOX A, BOX B and hematinic acid in a biological sample based on high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The present invention utilizes high performance liquid chromatography-triple quadrupole mass spectrometry technology to detect the contents of bilirubin oxidation products BOX A, BOX B and hematinic acid in the sample. The samples were deproteinized, analyzed by LC-MS/MS instrument in MRM mode, and quantified by external standard method. The method of the invention has the advantages of simplicity and rapidity, low detection limit, good stability, high detection sensitivity and good repeatability, is used for the determination of the oxidation product of bilirubin in biological samples, and can meet the detection requirements of target substances in biological samples , has high sensitivity, high selectivity, and strong anti-interference ability, and can be an effective tool for qualitative and quantitative analysis of bilirubin oxidation products.

Description

Detection and analysis method of bilirubin degradation products in biological samples based on LC-MS/MS

technical field

The invention relates to a method for detecting and analyzing biological samples, in particular to a method for detecting and analyzing degradation products of bilirubin in cerebrospinal fluid, which is applied to the technical field of target compound detection of biological samples.

Background technique

Stroke, also known as stroke, is an acute cerebrovascular disease characterized by high morbidity, high disability, high mortality, and high recurrence rate. Stroke includes ischemic stroke and hemorrhagic stroke, among which hemorrhagic stroke is caused by the rupture and hemorrhage of cerebral arteries. Subarachnoid hemorrhage is a type of hemorrhagic stroke, and its mortality rate is higher than the average mortality rate of stroke.

Studies have shown that the lysis of red blood cells after subarachnoid hemorrhage is the main reason that affects the recovery of patients. The red blood cells in the blood are lysed to release hemoglobin, and the heme in hemoglobin is further degraded into biliverdin, which is reduced to bilirubin under the action of biliverdin reductase. Bilirubin, as a reducing agent, can scavenge reactive oxygen species, and because of its antioxidant properties, it can be used as a protective agent when cells undergo oxidative stress. Studies have shown that cerebral hemorrhage can cause strong oxidative stress, release reactive oxygen species, and bilirubin acts as a reducing agent, which is oxidized to small molecular _compounds after encountering H2O2, among which BOX _A

(2-(3-ethenyl-1,5-dihydro-4-methyl-5-oxo-2H-pyrrol-2-ylidene)acetamide, bilirubin oxidizedA, CAS#329314-76-3), BOX B

(2-(4-ethenyl-1,5-dihydro-3-methyl-5-oxo-2H-pyrrol-2-ylidene)acetamide, bilirubin oxidizedB, CAS#329314-75-2) and hematinic acid (2,5 -dihydro-4-methyl-2,5-dioxo-1H-pyrrole- ₃ -propanoic acid, CAS# _487-65-0 ) is a reported degradation product of bilirubin under H2O2 oxidation. It has been experimentally verified that BOXA and BOX B can cause severe and persistent vasospasm of cerebral blood vessels in rats, which has important physiological significance. To study the contribution of bilirubin oxidation products to the complications of cerebral hemorrhage, the most direct and effective method is to detect the concentration of each compound in clinical samples, and to evaluate the relationship between the concentration of each compound and the disease. But so far, there is still a lack of detection methods with high sensitivity and high selectivity.

SUMMARY OF THE INVENTION

In order to solve the problems of the prior art, the object of the present invention is to overcome the deficiencies of the prior art, and to provide a method for detecting and analyzing the degradation products of bilirubin in biological samples based on LC-MS/MS, by using liquid chromatography- A tandem triple quadrupole mass spectrometer was used to establish the qualitative and quantitative methods for three compounds of bilirubin oxidation products, BOX A, BOX B and hematinic acid, and to optimize the instrumental analysis methods using the standard products of the three compounds, including liquid chromatography. The conditions, mass spectrometry detection conditions and mass spectrometry parameters are optimized to meet the detection requirements of accurate, rapid and high sensitivity determination of the three compounds.

In order to achieve the above-mentioned purpose of invention and creation, the present invention adopts the following technical solutions:

A detection and analysis method for bilirubin degradation products in biological samples based on LC-MS/MS, the detection and analysis steps include selecting liquid phase conditions, setting mass spectrometry conditions, preparing a standard solution, optimizing an analysis method, drawing a standard curve, a sample Pretreatment and qualitative and quantitative detection of products in cerebrospinal fluid; through the detection and analysis method, the three compounds of BOX A, BOX B and hematinic acid in cerebrospinal fluid are rapidly and accurately determined, and at the same time, the detection and analysis method is also used to measure cell lysis Fluid and blood sample information.

As a preferred technical solution of the present invention, a method for detecting and analyzing degradation products of bilirubin in biological samples based on LC-MS/MS includes the following steps:

S1: Select the liquid chromatography column and set the mass spectrometry conditions and parameters:

a. Set the HPLC conditions:

Chromatographic column: Agilent Poroshell EC-C18 column, 2.1×100mm, 2.7μm; column temperature: 30°C; set mobile phase and mobile phase gradient, wherein, water phase A: water and 0.1% (v/v) formic acid, Organic phase B: methanol and 0.1% (v/v) formic acid, methanol, acetonitrile or acetonitrile and 0.1% (v/v) formic acid, the flow rate is 0.3 mL/min, and the LC-MS/MS injection volume is set to 1-20 μL;

b. Set the mass spectrometry conditions:

Select the electrospray ion source (ESI), and select the appropriate ion scan mode according to the response of the compound;

c. Set the qualitative and quantitative detection mode of mass spectrometry:

Multiple reaction monitoring method (MRM), setting the parameters of multiple reaction monitoring mode;

S2: Prepare standard solution:

a. Weigh 2mg of BOX A, BOX B and hematinic acid and dissolve them in 1.1mL of water to prepare a 10mM stock solution;

b. Dilute with water to prepare a 500 μM molar concentration of hematinic acid solution, 10 μM BOXA and BOX B, and store in a refrigerator at 4°C away from light for later use;

S3: using a standard solution to optimize the instrument analysis method to obtain an optimized LC-MS/MS quantitative analysis method, wherein the instrument is a liquid chromatography-tandem triple quadrupole mass spectrometer;

S4: Plot the standard curve:

The hematinic acid standard stock solution prepared in the step S2 was diluted in proportion to prepare a hematinic acid standard solution with molar concentrations of 1, 5, 10, 50, 100, 150 and 200 μM, respectively. The standard stock solution was diluted proportionally to BOX A and BOX B standard solutions with molar concentrations of 5, 10, 50, 100, 200, 400, 600, 800, 1000 nM, and the injection volume was 1 μL; Standard curve, in which the peak area corresponds to the y-coordinate parameter on the ordinate, and the molar amount of the target corresponds to the abscissa-coordinate x parameter;

S5: Sample pretreatment:

Add 9 times the volume of ice ethanol to the cerebrospinal fluid sample, put it on ice for 30-60min, centrifuge at 15000rpm for 15-45min, take the supernatant; use a centrifugal concentrator to concentrate the supernatant to dryness and redissolve; Filter membrane, test the filtrate on the machine;

S6: Quantification by external standard method:

After pre-processing the sample in step S5, according to the drawn chromatogram, the peak area of each compound in the cerebrospinal fluid is calculated, and by obtaining the standard curve in step S4, BOX A and BOX B in the sample solution are calculated. and the concentration of hematinic acid, the linear relationship between BOX A and BOX B in the range of 5-1000fmol, and the concentration of hematinic acid in the range of 1-200pmol, and calculate the limit of detection (LOD) and limit of quantification (LOQ) of the instrument. ) and test repeatability information, while calculating intra-day and inter-day precision.

As a preferred technical solution of the present invention, in step a of step S1, the selected LC-MS/MS instruments are Agilent1290 high performance liquid chromatography system and AB SCIEX 4500 triple quadrupole mass spectrometer system.

As a preferred technical solution of the present invention, in step b of step S1, the mass spectrometry conditions are set as follows:

Select the electrospray ion source (ESI), and according to the ion response, BOX A and BOX B are scanned in positive ion mode, and hematinic acid is scanned in negative ion mode, using semi-automatic injection mode, with a flow rate of 7-20 μL/min to 300 μg/L The standard stock solution was injected into the ion source respectively, the corresponding parent ion peak was selected, and the secondary mass spectrometry was performed on the daughter ion to obtain the fragment ion information, and the MRM scanning method and parameters were established.

As a preferred technical solution of the present invention, the standard stock solution directly injected into the ion source is prepared with a mixed solvent of 50% (v/v) methanol and 50% (v/v) water.

As a preferred technical solution of the present invention, based on the LC-MS/MS detection and analysis method for the degradation products of bilirubin in cerebrospinal fluid, when establishing the MRM scanning method and parameters, the monitoring ion parameters of each compound are set, including parent ion, product ion, Cluster voltage (DP), collision energy (CE).

As a preferred technical solution of the present invention, in step c of step S1, other conditions for the MRM detection mode analysis of the LC-MS/MS are: ion source temperature 500°C, ion spray voltage 5500V, collision gas medium, The interface is heated on, the collision chamber inlet voltage (EP) is 10V, the collision chamber outlet voltage (CXP) is 11V, the air curtain air pressure is 40 psi, the spray air pressure is 50 psi, and the auxiliary heating air pressure is 50 psi.

As a preferred technical solution of the present invention, in the step S5, the centrifugation time is 20-60 min.

As a preferred technical solution of the present invention, in the step S6, the limit of detection (LOD) of the instrument is calculated with the signal-to-noise ratio S/N=3, and the limit of detection (LOQ) of the instrument is calculated with S/N=10, Assay repeatability is expressed as RSD % of standard peak area.

Further, the optimization of the instrument analysis method in the step S3 includes the optimization of liquid chromatography conditions, the optimization of mass spectrometry detection conditions, and the optimization of mass spectrometry parameters, so that the mass spectrometry can be accurately qualitative and the quantitative sensitivity is optimized.

Further, the amount of cerebrospinal fluid processed in the above step S5 is not limited, and the redissolved volume is not limited.

Further, in addition to the cerebrospinal fluid, the biological sample processed in the above step S5 can also be other biological samples.

Further, the intra-day and inter-day precisions in the above step S6 were measured with 1 μM BOX A and BOX B solutions and 200 μM hematinic acid solution for 3 consecutive days, three times a day.

Compared with the prior art, the present invention has the following obvious outstanding substantive features and significant advantages:

1. The present invention optimizes the instrumental analysis method by utilizing the standard products of the three compounds, including the optimization of liquid chromatography conditions, the optimization of mass spectrometry detection conditions and the optimization of mass spectrometry parameters, so that mass spectrometry can be accurately qualitative and quantitative sensitivity is optimized;

2. The present invention adopts LC-MS/MS and uses high performance liquid chromatography-tandem triple quadrupole mass spectrometer to establish a qualitative and quantitative method for three oxidation products of bilirubin in cerebrospinal fluid, and the method has the advantages of simplicity. The advantages of rapidity, small sample volume, low detection limit, good repeatability and high sensitivity;

3. The method of the invention is simple and easy to implement, low in cost, and suitable for popularization and use.

Description of drawings

Fig. 1 is the chromatogram of the hematinic acid standard solution of the second embodiment of the present invention.

Fig. 2 is the chromatogram of the BOX A standard solution of the second embodiment of the present invention.

Fig. 3 is the chromatogram of the BOX B standard solution of the second embodiment of the present invention.

Fig. 4 is the chromatogram of the negative ion mode detection in the cerebrospinal fluid according to the second embodiment of the present invention (detection of hematinic acid).

Fig. 5 is a chromatogram of positive ion mode detection in cerebrospinal fluid according to the second embodiment of the present invention (BOX A and BOXB are detected).

Detailed ways

The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, the detection of BOX A, BOX B and hemanitic acid in cerebrospinal fluid, blood and other biological samples by this method in the art all belong to the protection scope of the present invention.

The above scheme will be further described below in conjunction with specific embodiments, and preferred embodiments of the present invention are described in detail as follows:

Example 1:

In this embodiment, a method for detecting and analyzing degradation products of bilirubin in biological samples based on LC-MS/MS is characterized in that, the detecting and analyzing steps include selecting liquid phase conditions, setting mass spectrometry conditions, and preparing standard solutions , optimize the analysis method, draw the standard curve, sample pretreatment and qualitative and quantitative detection of the products in the cerebrospinal fluid; through the detection and analysis method, the three compounds of BOX A, BOX B and hematinic acid in the cerebrospinal fluid are quickly and accurately determined. The assay methods described above are also used to measure cell lysate and blood sample information.

The method of this example uses LC-MS/MS to establish a method for determining the contents of BOX A, BOX B and hemaniticacid in cerebrospinal fluid. LC-MS/MS has high sensitivity, high selectivity, and strong anti-interference ability, and can be an effective tool for qualitative and quantitative analysis of bilirubin oxidation products.

Embodiment 2:

This embodiment is basically the same as the first embodiment, and the special features are:

In this embodiment, Agilent 1290 high performance liquid chromatography system and AB SCIEX 4500 triple quadrupole mass spectrometer system are selected as the instruments. A method for detecting and analyzing degradation products of bilirubin in cerebrospinal fluid based on LC-MS/MS, comprising the following steps:

S1: Select the liquid chromatography column and set the mass spectrometry conditions and parameters:

a. Set the HPLC conditions:

Chromatographic column: Agilent Poroshell EC-C18 column, 2.1×100mm, 2.7μm; column temperature: 30°C; set mobile phase and mobile phase gradient, wherein, water phase A: water and 0.1% (v/v) formic acid, Organic phase B: methanol and 0.1% (v/v) formic acid, the flow rate is 0.3 mL/min, and the LC-MS/MS injection volume is set to 1-20 μL;

Table 1 Mobile phase gradient parameters

b. Set the mass spectrometry conditions, and set the mass spectrometry qualitative and quantitative detection mode:

Select the electrospray ion source (ESI), and select the appropriate ion scan mode according to the response of the compound; use the multiple reaction monitoring method (MRM) to set the parameters of the multiple reaction monitoring mode; the details are as follows:

Select the electrospray ion source, BOX A and BOX B are scanned in positive ion mode, hematinic acid is scanned in negative ion mode, semi-automatic injection mode is used, and 300 μg/L of standard stock solution is injected into the ion source respectively at a flow rate of 7 μL/min; The corresponding parent ion peaks were analyzed by secondary mass spectrometry to obtain fragment ion information, and the MRM mass spectrometry detection method of the three compounds was established;

Table 2 MRM mass spectrometry detection parameters of three compounds

During the mass spectrometry analysis described in the above scheme, the ion source temperature was 500°C, the ion spray voltage was 5500V, the collision gas was medium, the interface heating was on, the EP was 10V, the CXP was 11V, the curtain gas pressure was 40 psi, the spray gas pressure was 50 psi, and the auxiliary gas pressure was 50 psi. Heating gas pressure 50psi;

S2: Prepare standard solution:

a. Weigh 2mg of BOX A, BOX B and hematinic acid and dissolve them in 1.1mL of water to prepare a 10mM stock solution;

b. Dilute with water to prepare a 500 μM molar concentration of hematinic acid solution, 10 μM BOXA and BOX B, and store in a refrigerator at 4°C away from light for later use;

S3: using a standard solution to optimize the instrument analysis method to obtain an optimized LC-MS/MS quantitative analysis method, wherein the instrument is a liquid chromatography-tandem triple quadrupole mass spectrometer;

S4: Plot the standard curve:

The hematinic acid standard stock solution prepared in the step S2 was diluted in proportion to prepare a hematinic acid standard solution with molar concentrations of 1, 5, 10, 50, 100, 150 and 200 μM, respectively. The standard stock solution was diluted proportionally to BOX A and BOX B standard solutions with molar concentrations of 5, 10, 50, 100, 200, 400, 600, 800, 1000 nM, and the injection volume was 1 μL; Standard curve, wherein the peak area corresponds to the y parameter on the ordinate, and the molar amount of the target corresponds to the x parameter on the abscissa; the obtained standard curve is shown in the results in Table 3.

Table 3 Results of LC-MS/MS quantitative analysis method for three compounds

S5: Sample pretreatment:

Take 5 mL of 6 cerebrospinal fluid samples, add 9 times the volume of ice-cold ethanol, place on ice for 30 min, centrifuge at 15,000 rpm for 30 min, take the supernatant, centrifuge and concentrate to 5 mL, and find that there is still protein, then add 9 times the volume of ethanol. Ice ethanol, placed on ice for 40min, centrifuged at 15000rpm for 30min, took the supernatant, centrifuged and concentrated to dryness, added 50μL of water to redissolve, and waited for LC-MS/MS determination;

S6: Quantification by external standard method:

After pre-processing the sample in step S5, according to the drawn chromatogram, the peak area of each compound in the cerebrospinal fluid is calculated, and by obtaining the standard curve in step S4, BOX A and BOX B in the sample solution are calculated. and the concentration of hematinic acid, 6 samples were detected by the MRM detection method of BOX A, BOX B and hematinic acid established on the LC-MS/MS instrument. The obtained peak area is converted to the corresponding working curve, and then multiplied by the concentration multiple (100 times) to obtain the concentration of each compound in the 6 samples; the LC-MS/MS spectrum of one sample is shown in Figure 4 and Figure 5 ; The detection results of 6 samples are summarized in Table 4;

The concentration of each product measured in the cerebrospinal fluid of table 4

Experimental test analysis:

For the technical effect of the method of the present embodiment, the energy-saving test and analysis, Fig. 1 is the chromatogram of the hematinicacid standard solution of the second embodiment of the present invention. Fig. 2 is the chromatogram of the BOX A standard solution of the second embodiment of the present invention. Fig. 3 is the chromatogram of the BOX B standard solution of the second embodiment of the present invention. The experimental verification is as follows:

1. Selection of mobile phase: In order to optimize the mobile phase conditions, in this experiment, BOX A and BOX B used ESI ⁺ mode, hematinic acid used ESI ^- mode, and acetonitrile-water, acetonitrile (containing 0.1% formic acid)-water (containing 0.1% formic acid) were used respectively. 0.1% formic acid), methanol-water, methanol (containing 0.1% formic acid)-water (containing 0.1% formic acid) were used as mobile phases to compare the separation effects of BOX A, BOX B and hematinicacid, and it was found that methanol-water was used as the mobile phase. The mass spectral response and resolution of the target peaks are better than those of acetonitrile-water solution, and the addition of formic acid can maintain the stability of the peak and maintain pH stability, which helps to prolong the life of the chromatographic column. Therefore, methanol (containing 0.1% formic acid) is selected. )-water (containing 0.1% formic acid) was the mobile phase.

2. Selection of elution program: Elution programs with different starting concentrations and different slopes were compared, and finally the elution program shown in Table 1 was selected.

The method of this embodiment uses high performance liquid chromatography-triple quadrupole mass spectrometry to detect the contents of bilirubin oxidation products BOX A, BOX B and hematinic acid in the sample. The samples were deproteinized, analyzed by LC-MS/MS instrument in MRM mode, and quantified by external standard method. The method of the invention has the advantages of simplicity and rapidity, low detection limit, good stability, high detection sensitivity and good repeatability, and can be used for the determination of bilirubin oxidation products BOX A, BOX B and hematinic acid in cerebrospinal fluid, and can meet the requirements of cerebrospinal fluid. detection requirements of the target. In this example, a method for determining the contents of BOX A, BOX B and hemanitic acid in cerebrospinal fluid is established by LC-MS/MS. LC-MS/MS has high sensitivity, high selectivity, and strong anti-interference ability, and can be an effective tool for qualitative and quantitative analysis of bilirubin oxidation products.

Embodiment three:

LC-MS/MS-based detection and analysis method for bilirubin degradation products in biological samples, in addition to cerebrospinal fluid detection, it can also detect BOX A, BOX B and hematinic acid in other biological samples such as blood and cell lysate. concentration.

Bilirubin oxidation products can cause cerebral vasospasm, suggesting that it may be physiologically toxic. There are a large number of red blood cells in the blood, which may produce BOX A, BOX B and hematinic acid when subjected to oxidative stress, and may cause other diseases when the levels are too high in the blood.

Using this detection method to detect the concentration of each compound in the blood sample specifically includes the following steps:

S1: Select the liquid chromatography column and set the mass spectrometry conditions and parameters:

a. High performance liquid chromatography conditions: chromatographic column: Agilent Poroshell EC-C18 column, 2.1×100mm, 2.7μm; column temperature: 30°C; set mobile phase and mobile phase gradient, wherein, water phase A: water and 0.1 % (v/v) formic acid, organic phase B: methanol and 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min;

b. Mass spectrometry conditions: select the electrospray ion source, scan BOX A and BOX B in positive ion mode, scan in negative ion mode for hematinic acid, and use semi-automatic injection mode to inject 300 μg/L of standard stock solution at a flow rate of 7 μL/min. Ion source; select the corresponding parent ion peak, perform secondary mass spectrometry analysis on its product ion, and obtain fragment ion information;

c. Set the qualitative and quantitative detection mode of mass spectrometry: MRM mode, set parameters: ion source temperature is 500℃, ion spray voltage is 5500V, collision gas is medium, interface heating is on, EP is 10V, CXP is 11V, and curtain gas pressure is 40psi, Spray gas pressure 50psi, auxiliary heating gas pressure 50psi;

S2: Prepare standard solution:

a. Weigh 2mg of BOX A, BOX B and hematinic acid and dissolve them in 1.1mL of water to prepare a 10mM stock solution;

b. Dilute with water to prepare a 500μM (91.5μg/mL) hematinic acid solution, 10μM (1.79μg/mL) BOX A and BOX B, and store in a refrigerator at 4°C in the dark for future use;

S3: using a standard solution to optimize the instrument analysis method to obtain an optimized LC-MS/MS quantitative analysis method, wherein the instrument is a liquid chromatography-tandem triple quadrupole mass spectrometer;

S4: Plot the standard curve:

Dilute the standard stock solution of hematinic acid in S2 in proportion to prepare a standard solution of hematinic acid with a molar concentration of 1, 5, 10, 50, 100, 150, 200 μM, and dilute the standard stock solutions of BOX A and BOX B to a molar concentration respectively. BOX A and BOX B standard solutions of 5, 10, 50, 100, 200, 400, 600, 800, 1000 nM, and the injection volume is 1 μL;

Draw a standard curve with the peak area (y) versus the molar amount of the target substance (x);

S5: Sample pretreatment:

After taking the serum from the fresh 4mL blood sample, add 9 times the volume of ice-cold ethanol, place on ice for 30min, centrifuge at 15000rpm for 30min, take the supernatant, and repeat this step until there is no protein precipitation in the supernatant;

The supernatant was concentrated to dryness with a centrifugal concentrator, and redissolved in 40 μL of water;

Pass the 0.22μm filter membrane, and test the filtrate on the machine;

S6: Quantification by external standard method: after the analysis in step S5, calculate the peak area of each compound in the serum according to the drawn chromatogram, and calculate the BOX A, BOX B and hematinic acid in the sample solution through the standard curve described in step S4 The concentration of BOX A and BOX B is in the range of 5-1000fmol, and the hematinic acid has a good linear relationship in the range of 1-200pmol. The LOD of the instrument is calculated by the signal-to-noise ratio S/N=3, and the signal-to-noise ratio S/N = 10 Calculate the LOQ of the instrument, and simultaneously use 1 μM BOX A and BOX B solutions, 200 μM hematinic acid solution for 3 consecutive days, and measure 3 times a day to obtain intra-day and inter-day precision.

The method of this embodiment is based on the method for determining the contents of three bilirubin oxidation products BOX A, BOX B and hematinic acid in biological samples by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The invention utilizes high performance liquid chromatography-triple quadrupole mass spectrometry technology to detect the contents of bilirubin oxidation products BOX A, BOX B and hematinic acid in the sample. The samples were deproteinized, analyzed by LC-MS/MS instrument in MRM mode, and quantified by external standard method. The method of this embodiment has the advantages of simplicity and rapidity, low detection limit, good stability, high detection sensitivity and good repeatability. Requirements for the detection of targets in cerebrospinal fluid.

The embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned embodiments, and various changes can also be made according to the purpose of the invention and creation of the present invention. Changes, modifications, substitutions, combinations or simplifications should be equivalent substitution methods, as long as they meet the purpose of the present invention, as long as they do not deviate from the LC-MS/MS-based LC-MS/MS of the present invention for degradation products of bilirubin in biological samples. The technical principle and inventive concept of the detection and analysis method all belong to the protection scope of the present invention.

Claims (9)

1. a detection analysis method based on LC-MS/MS to the degradation product of bilirubin in biological sample, it is characterized in that, detection analysis step comprises selecting liquid phase condition, setting mass spectrometry condition, preparing standard solution, optimizing analysis method , draw standard curve, sample pretreatment and qualitative and quantitative detection of products in cerebrospinal fluid; through the detection and analysis method, the three compounds of BOX A, BOX B and hematinic acid in cerebrospinal fluid are quickly and accurately determined. At the same time, the detection and analysis method Also used to measure cell lysate and blood sample information. 2. the method for detecting and analyzing the degradation product of bilirubin in biological samples based on LC-MS/MS according to claim 1, is characterized in that: comprising the steps: S1: Select the liquid chromatography column and set the mass spectrometry conditions and parameters: a. Set the HPLC conditions: Chromatographic column: Agilent Poroshell EC-C18 column, 2.1×100mm, 2.7μm; column temperature: 30°C; set mobile phase and mobile phase gradient, wherein, water phase A: water and 0.1% (v/v) formic acid, Organic phase B: methanol and 0.1% (v/v) formic acid, methanol, acetonitrile or acetonitrile and 0.1% (v/v) formic acid, the flow rate is 0.3 mL/min, and the LC-MS/MS injection volume is set to 1-20 μL; b. Set the mass spectrometry conditions: Select the electrospray ion source (ESI), and select the appropriate ion scan mode according to the response of the compound; c. Set the qualitative and quantitative detection mode of mass spectrometry: Multiple reaction monitoring method (MRM), setting the parameters of multiple reaction monitoring mode; S2: Prepare standard solution: a. Weigh 2mg of BOX A, BOX B and hematinic acid and dissolve them in 1.1mL of water to prepare a 10mM stock solution; b. Dilute with water to prepare a 500μM molar concentration of hematinic acid solution, 10μM BOX A and BOX B respectively, and store in a refrigerator at 4°C away from light for later use; S3: using a standard solution to optimize the instrument analysis method to obtain an optimized LC-MS/MS quantitative analysis method, wherein the instrument is a liquid chromatography-tandem triple quadrupole mass spectrometer; S4: Plot the standard curve: The hematinic acid standard stock solution prepared in the step S2 was diluted in proportion to prepare a hematinic acid standard solution with molar concentrations of 1, 5, 10, 50, 100, 150 and 200 μM, respectively. The standard stock solution was diluted proportionally to BOX A and BOX B standard solutions with molar concentrations of 5, 10, 50, 100, 200, 400, 600, 800, 1000 nM, and the injection volume was 1 μL; Standard curve, in which the peak area corresponds to the y-coordinate parameter on the ordinate, and the molar amount of the target corresponds to the abscissa-coordinate x parameter; S5: Sample pretreatment: Add 9 times the volume of ice ethanol to the cerebrospinal fluid sample, put it on ice for 30-60min, centrifuge at 15000rpm for 15-45min, take the supernatant; use a centrifugal concentrator to concentrate the supernatant to dryness and redissolve; Filter membrane, test the filtrate on the machine; S6: Quantification by external standard method: After pre-processing the sample in step S5, according to the drawn chromatogram, the peak area of each compound in the cerebrospinal fluid is calculated, and by obtaining the standard curve in step S4, BOX A and BOX B in the sample solution are calculated. and the concentration of hematinic acid, the linear relationship between BOX A and BOX B in the range of 5-1000fmol, and the concentration of hematinic acid in the range of 1-200pmol, and calculate the limit of detection (LOD) and limit of quantification (LOQ) of the instrument. ) and test repeatability information, while calculating intra-day and inter-day precision. 3. The method for detecting and analyzing the degradation products of bilirubin in biological samples based on LC-MS/MS according to claim 2, characterized in that: in the step a of the step S1, the selected LC-MS/MS The instruments are Agilent 1290 high performance liquid chromatography system and AB SCIEX 4500 triple quadrupole mass spectrometer system. 4. The method for detecting and analyzing the degradation products of bilirubin in biological samples based on LC-MS/MS according to claim 2, characterized in that: in the step b of the step S1, the mass spectrometry conditions are set as follows: Select the electrospray ion source (ESI), and according to the ion response, BOX A and BOX B are scanned in positive ion mode, and hematinic acid is scanned in negative ion mode, using semi-automatic injection mode, with a flow rate of 7-20 μL/min to 300 μg/L The standard stock solution was injected into the ion source respectively, the corresponding parent ion peak was selected, and the secondary mass spectrometry was performed on the daughter ion to obtain the fragment ion information, and the MRM scanning method and parameters were established. 5. The method for detecting and analyzing the degradation products of bilirubin in biological samples based on LC-MS/MS according to claim 4, characterized in that: the standard stock solution directly injected into the ion source uses 50% (v/v) methanol It is formulated with 50% (v/v) water mixed solvent. 6. The method for detecting and analyzing the degradation products of bilirubin in biological samples based on LC-MS/MS according to claim 4, characterized in that: when establishing the MRM scanning method and parameters, the monitoring ion parameters of each compound are set to include parent ions , product ions, declustering potential (DP), collision energy (CE). 7. The method for detecting and analyzing degradation products of bilirubin in biological samples based on LC-MS/MS according to claim 2, wherein in step c of step S1, the LC-MS/MS Other conditions for MRM detection mode analysis are: ion source temperature 500°C, ion spray voltage 5500V, collision gas medium, interface heating on, collision chamber inlet voltage (EP) 10V, collision chamber outlet voltage (CXP) 11V, and curtain gas pressure 40 psi, spray gas pressure 50 psi, auxiliary heating gas pressure 50 psi. 8 . The method for detecting and analyzing the degradation products of bilirubin in biological samples based on LC-MS/MS according to claim 2 , wherein, in the step S5 , the centrifugation time is 20-60 min. 9 . 9. The method for detecting and analyzing the degradation products of bilirubin in biological samples based on LC-MS/MS according to claim 2, wherein in the step S6, the signal-to-noise ratio S/N=3 is used to calculate The limit of detection (LOD) of the instrument was taken as S/N=10 as the limit of detection of quantification (LOQ) of the instrument, and the detection repeatability was expressed as the RSD% of the peak area of the standard product.

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